JP4756601B2 - Electrocatalyst and enzyme electrode - Google Patents

Description

  The present invention relates to an electrode catalyst and an enzyme electrode, and more particularly to an enzyme electrode used as an electrode of various electrochemical devices such as a biosensor and a fuel cell, and an electrode catalyst used for the enzyme electrode.

An enzyme is a biocatalyst that promotes a chemical reaction (metabolism) in a living body, and has a protein as a basic structure. Some enzymes consist only of proteins, but many of them require components (cofactors) other than proteins in order to express catalytic activity or enhance catalytic activity.
The enzyme
(1) Catalytic activity under mild conditions around normal temperature and normal pressure,
(2) Combined with “substrate specificity” that acts only on specific substrates (substances that are affected by enzymes) and “reaction specificity” that exhibits a catalytic action on specific chemical reactions and does not cause side reactions. ,
There is a feature.
Among enzymes, an enzyme that catalyzes redox in a living body is called “oxidoreductase”. In addition, an enzyme that oxidizes a substrate using oxygen as an electron acceptor is particularly referred to as an “oxidase”, and an enzyme that reduces a substrate is particularly referred to as a “reductase”. When a certain oxidoreductase is immobilized on the electrode surface, only a specific redox reaction proceeds selectively on the electrode due to the catalytic action of the enzyme, and the change of the substance due to the redox reaction is converted into an electrical signal by the electrode. Can do. Such an electrode is called an “enzyme electrode” and is used for various biosensors, fuel cells, and the like.

In order to use an enzyme as an electrocatalyst, it is necessary to fix it on a suitable carrier surface. However, since the enzyme is generally water-soluble, there is a problem that the enzyme tends to flow out during use.
In addition, the enzyme has a site (active center) where the substrate specifically binds and is catalyzed. Since the active center is often buried deep in a protein having a complicated three-dimensional structure, it is difficult to exchange electrons directly with the electrode. In such a case, a low molecular weight substance that normally enters the active site of the enzyme, transfers the enzyme and electrons, and carries the electrons to the electrode is used in combination. Such low molecular weight substances are called “mediators”. However, since the electron transfer rate between the enzyme / mediator depends on the molecular motion of each, it is not always sufficient, which may be a factor limiting the current density of the enzyme electrode.

In order to solve this problem, various proposals have heretofore been made.
For example, Patent Document 1 discloses a nitrogen-containing carbon-based composite including a porous body made of a nitrogen-containing carbon-based material having a skeleton formed of carbon atoms and nitrogen atoms, and an oxidoreductase supported on the porous body. A material is disclosed.
In the same document,
(1) Since nitrogen-containing carbon-based materials are interspersed with polar points on the surface of pores, when an oxidoreductase is supported on this, new bonds such as hydrogen bonds are formed with hydrophilic groups on the protein surface, The bond between the protein and the carrier is strengthened, and
(2) As oxidoreductases to be supported on a carrier, laccase, diaphorase, lipoxyamide dehydrogenase, alcohol dehydrogenase, glucose oxidase (including oxidase with other sugar as substrate), glucose dehydrogenase (dehydrogenase with other sugar as substrate) Point)
Is described.

Patent Document 2 discloses an enzyme electrode having a conductive member, an enzyme, and first and second mediators, wherein the first mediator and the second mediator are attached to the conductive member by a carrier. An enzyme electrode in which the first mediator and the second mediator are immobilized and have different redox potentials is disclosed.
In the same document,
(1) By adopting such a configuration, the enzyme carrying density per effective surface area of the conductive member can be increased,
(2) By further using a second mediator that performs charge transport between the first mediator and the conductive member in addition to the first mediator capable of performing high-speed electron transfer with the enzyme. And (3) as an enzyme, glucose oxidase, galactose oxidase, bilirubin oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, Ascorbate oxidase, cytochrome oxidase, alcohol dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase, fructose dehydrogenase, sorbitol Hydrogenase, lactate dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase, amino acid dehydrogenase, glyceryl aldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diaphorase, catalase, peroxidase, glutathione reductase, NADH-cytochrome b5 reductase, NADPH-adrenoxin reductase, cytochrome b5 reductase, adrenodoxin reductase, point that nitrate reductase is mentioned,
Is described.

JP 2005-343775 A JP 2006-058289 A

In general, the oxidoreductase has a site (electron transfer gate) for exchanging electrons with the active center in addition to the active center that binds to the substrate. Since electron transfer to an enzyme in a living body is usually mediated by a mediator, the electron transfer gate of the enzyme is often buried inside a protein pocket. When the enzyme in such a state is immobilized on the electrode surface, the distance between the electron transfer gate and the electrode surface inevitably increases. Since the common logarithm of the electron transfer speed becomes slower in proportion to the distance, a conventionally known enzyme cannot obtain a high electron transfer efficiency.
In order to solve this problem, it is conceivable to use a mediator in combination as disclosed in Patent Document 2. However, there is a limit in improving the electron transfer efficiency only by the method using the mediator together. In addition, there has been no example in which an enzyme suitable for an enzyme electrode capable of obtaining a sufficient current density has been reported.

  The problem to be solved by the present invention is to provide a novel electrode catalyst comprising an oxidase having high electron transfer efficiency, and an enzyme electrode using the same.

In order to solve the above-described problems, the electrode catalyst according to the present invention is made of Escherichia coli-derived CueO.
The enzyme electrode according to the present invention includes a carbon porous body and an electrode catalyst made of Escherichia coli-derived CueO supported on the surface of the carbon porous body.

When CueO, which is a kind of multi-copper oxidase, is used as a catalyst for an enzyme electrode, a higher current density can be obtained than conventional oxidoreductases.
This is considered to be due to the following reason.
(1) Since CueO separates the active center that catalyzes the oxidation reaction and the electron transfer gate in the enzyme molecule, even if the enzyme is immobilized on the electrode solid surface, the catalytic reaction and the electron transfer can be achieved simultaneously.
(2) Since CueO has more electron transfer gates on the surface of the molecule, the distance from the electrode is reduced in the state of being fixed on the solid surface, and the efficiency of electron transfer is improved.

Hereinafter, an embodiment of the present invention will be described in detail.
The electrode catalyst according to the present invention is made of CueO.
An oxidoreductase is an enzyme that catalyzes a redox reaction, and has an active center and an electron transfer gate in the three-dimensional structure of a protein. “Active center” refers to a site where a substrate specifically binds and is catalyzed. “Electron transfer gate” refers to a site for transferring electrons to and from the active center.
An oxidoreductase is roughly classified into an oxidase that oxidizes a substrate and a reductase that reduces a substrate. Which one is used as an electrode catalyst enzyme depends on the polarity of the electrode. That is, when an enzyme is used as a material on the positive electrode side, an oxidase that can receive electrons by the reaction (in other words, can catalyze a reaction that generates water using protons and oxygen as substrates) is used. On the other hand, when an enzyme is used as the negative electrode material, a reductase that releases electrons by reaction is used.

In addition, oxidoreductase is a macromolecule with a unique three-dimensional structure, and the active center and electron transfer gate are usually buried in the back of the three-dimensional structure opening (indentation, crack). . In this case, the active center and the electron transfer gate may be in the same opening or in different openings.
Among these, the oxidoreductase having the first opening including the electron transfer gate and the second opening including the active center is such that when the enzyme catalyzes the substrate, the movement of the substrate is the movement of the electron. There is no interference. Therefore, the electron transfer efficiency between the enzyme / electrode is improved as compared with an enzyme including an active center and an electron transfer gate in the same opening.
In particular, the oxidoreductase in which the first opening including the electron transfer gate is opposite to the second opening including the active center is disposed between the electrode and the electron transfer gate by bringing the first opening close to the electrode surface. Can be transferred very smoothly. Therefore, the efficiency of electron transfer between the enzyme / electrode is further improved.
Here, “the first opening is opposite to the second opening” means that the normal direction of the surface having the first opening and the normal direction of the surface having the second opening are The angle formed is greater than 90 °. The angle formed by the two normals is preferably larger, and the closer to 180 °, the better.

Among the oxidoreductases, multi-copper oxidase catalyzes a reaction that generates four electrons of molecular oxygen using electrons taken out from a substrate to generate water. Multi-copper oxidase has mononuclear blue copper (type ICu) as an electron transfer gate and trinuclear copper cluster (type IICu, type IIICu) as an active center. Mononuclear blue copper and trinuclear copper cluster are Each is inside a different opening. Moreover, the two openings are respectively on the front and back surfaces of the three-dimensional structure formed by the protein. Therefore, multi-copper oxidase or a modified enzyme obtained by modifying this by gene manipulation is particularly suitable as a catalyst for the positive electrode of an enzyme electrode used in various electrochemical devices such as biosensors and fuel cells.
Specific examples of the multi-copper oxidase include laccase, bilirubin oxidase, ascorbate oxidase, ceruloplasmin, CueO, and endospore coat protein CotA of Bacillus bacteria. Among these, CueO (particularly, copper metabolism-related oxidase CueO of E. coli (CueO derived from E. coli)) has a higher current density than conventional oxidoreductases when used as an electrode catalyst for enzyme electrodes. It is done.

“CueO” refers to a type of multi-copper oxidase collected from various strains. Further, “E. coli-derived CueO” refers to CueO collected from E. coli. The amino acid sequence (swissprot access: Q8X947) of natural mature CueO is shown in SEQ ID NO: 1 in the sequence listing.
CueO may be natural CueO collected directly from a strain, or modified CueO in which a part of the amino acid sequence of natural CueO is changed or deleted by genetic manipulation within a range that does not lose catalytic activity and electron transfer ability. It may be.

Next, the enzyme electrode according to the present invention will be described.
The enzyme electrode according to the present invention includes a carbon porous body and an electrode catalyst made of CueO supported on the surface of the carbon porous body. CueO is particularly preferably E. coli-derived CueO. Among these, CueO and Escherichia coli-derived CueO are the same as described above, and thus description thereof is omitted.

  A carbon porous body is used for the carrier supporting the enzyme. The carbon porous body not only facilitates electron transfer between the enzyme and the electrode, but also can physically support the enzyme in the pore when the pore size is appropriate, and also suppresses enzyme deactivation. Since it can be used, it is particularly suitable as a carrier.

In order to sufficiently improve the stability and activity of the enzyme that is the supporting component, the porous carbon body has the following [1. ] To [4. It is preferable to have one or more of the following conditions.
[1. Average pore diameter]
The carbon porous body preferably has an average pore diameter of 2 to 50 nm. The average pore diameter is more preferably 2 to 20 nm. When the average pore diameter is less than 2 nm, the pore size is often smaller than the enzyme size, and the adsorptivity is reduced. On the other hand, when the average pore diameter exceeds 50 nm, the specific surface area is reduced and the adsorptivity is reduced. Further, if the average pore diameter exceeds 20 nm, there is a risk that inconvenience is likely to occur when a part of the enzyme is supported.
The average pore diameter of the carbon porous body is preferably equal to or larger than the molecular diameter of the enzyme. The average pore diameter is more preferably about 1 to 1.25 times the molecular diameter of the enzyme. When the average pore diameter of the carbon porous body is in the above range, the enzyme is easily fixed in the pores. When the enzyme is immobilized in the pore, the outer wall of the pore suppresses the structural change of the enzyme due to heat, so that the inactivation of the enzyme due to heat can be suppressed, and the thermal stability is improved.
The average pore diameter means a pore diameter value at the top of the distribution peak in a pore diameter distribution obtained from a nitrogen adsorption isotherm described later.

[2. Pore size distribution]
The carbon porous body preferably has a pore volume of 60% or more in the range of ± 25% of the average pore diameter based on the total pore volume in the range of the pore diameter distribution range of 2 to 100 nm. If the uniformity of the pore diameter is worse than this, the number of pores other than the optimum pore diameter for supporting the enzyme increases, and the stability and activity of the enzyme may be insufficient.

[3. Specific surface area]
The carbon porous body preferably has a specific surface area of 100 m ² / g or more. The specific surface area is more preferably 500 to 1000 m ² / g. When the specific surface area of the carbon porous body is less than 100 m ² / g, the contact area with the enzyme decreases and the pores that take in the enzyme decrease, and the adsorptivity of the enzyme decreases.

[4. Pore volume]
The pore volume of the carbon porous body is not particularly limited because it varies depending on the specific surface area and the average pore diameter, but is preferably 0.1 to 50 ml / g. The pore volume is more preferably 0.2 to 2.5 ml / g.
The total volume of pores having a pore diameter equal to or larger than the molecular diameter of the enzyme among the pores of the carbon porous body is preferably equal to or greater than the total volume of the supported enzyme.

The specific surface area and pore volume of the carbon porous body can be determined by the general volumetric method described below.
That is, the carbon porous body is put in a container and cooled to a liquid nitrogen temperature (−198 ° C.), nitrogen gas is introduced into the container, and the adsorption amount is obtained by a volume method. Next, the pressure of the introduced nitrogen gas is gradually increased, and the nitrogen gas adsorption amount with respect to each equilibrium pressure is plotted to obtain a nitrogen adsorption / desorption isotherm. Using this nitrogen adsorption / desorption isotherm, the specific surface area and pore volume can be calculated by the SPE (Subtracting Pore Effect) method (K.Kaneko, C.Ishii, M.Ruike, H.Kuwabara, Carbon 30, 1075, 1986). The SPE method is a method in which micropore analysis is performed by αs-plot method, t-plot method, etc., and the effect of the strong potential field of micropores is removed to calculate the specific surface area and the like. This method is more accurate than the BET method in calculating the specific surface area of the body.
In addition, the pore size distribution and the pore size value of the distribution peak top can be obtained by BJH analysis from the obtained nitrogen adsorption / desorption isotherm (Barret EP, Joyner LG, Halenda PH, Journal of American Chemical Society 73, 373, 1951).

Specific examples of the carbon porous body that satisfies the above-described conditions include the following.
[1. Mesoporous carbon particles]
A first specific example of the carbon porous body is mesoporous carbon particles. “Mesoporous carbon particles” is a porous carbon material in which meso-sized (2 to 10 nm) pores are regularly arranged, and is 2 to 2 with respect to the total pore volume (S ₀ ) in a pore diameter distribution region of 2 to 100 nm. The ratio (= S × 100 / S ₀ ) of the total pore volume (S) in the pore diameter distribution region of 10 nm is 80% or more. Such pore size distribution can be measured by the XRD method and the nitrogen adsorption method.

[2. Carbon gel]
A second specific example of the carbon porous body is carbon gel. The “carbon gel” is a porous carbon material in which meso-sized pores are irregularly arranged and satisfies the following conditions (a) and (b).
(A) No X-ray diffraction peak is observed in the scan region 2θ = 0.5 to 10 ° (CuKα ray).
(B) In the pore size distribution calculated from the adsorption / desorption isotherm,
When the pore diameter value (d) at the distribution peak top is in the range of 2 nm or more and less than 10 nm, 60% or more of the total pore volume in the pore diameter region of d ± 2 nm with respect to the pore diameter value (d). Is included,
When the pore diameter value (D) at the distribution peak top is in the range of 10 nm to 50 nm, (0.75 × D) to (1.25 × D) nm with respect to the pore diameter value (D). 60% or more of the total pore volume is included in the pore diameter region.

Condition (A) represents the presence or absence of a regular arrangement of mesopores. The X-ray diffraction peak means that there is a periodic structure having a d value corresponding to the peak angle in the sample. Therefore, the carbon porous body in which one or more peaks are observed in the scan region 2θ = 0.5 to 10 ° (CuKα ray) has so-called pores regularly arranged with a period of 0.9 to 17.7 nm. Mesoporous carbon (MPC).
On the other hand, in the carbon gel, no X-ray diffraction peak is observed in the scan region 2θ = 0.5 to 10 ° (CuKα ray). This indicates that the pores in the carbon gel do not have a periodic arrangement structure. Carbon gel has a three-dimensional network structure in which pores are connected to each other. When carbon gel is used as a carrier, the details of the reason are unclear, but the stability and activity of the enzyme are improved as compared with the case where mesoporous carbon is used.

  In the X-ray diffraction measurement (XRD), a peak intensity ratio of less than 3 is not recognized as an X-ray diffraction peak. That is, “no X-ray diffraction peak is observed in the scan region 2θ = 0.5 to 10 ° (CuKα ray)” means that the background noise intensity in the scan region 2θ = 0.5 to 10 ° (CuKα ray). This means that no X-ray diffraction peak having a peak intensity ratio of 3 or more is observed.

  The condition (b) represents the uniformity of the pore diameter. When the pore distribution is in the above range, the amount of pores having the optimum pore diameter for supporting the enzyme increases, so that the stability and activity of the enzyme are improved.

It is preferable that the carbon gel further includes the following condition (c) in addition to the above (a) and (b).
(C) In the pore size distribution, the carbon gel preferably has a pore size value at the distribution peak top of 2 to 20 nm.
If the pore diameter value of the carbon gel is less than 2 nm, the pore size is often smaller than the enzyme size, so that the adsorptivity decreases. On the other hand, when the pore diameter value exceeds 50 nm, the specific surface area is reduced and the adsorptivity is reduced. Moreover, when the pore size value of the distribution peak top exceeds 20 nm, there is a possibility that inconvenience may occur when the enzyme is supported.

[3. Nitrogen-containing carbon gel]
A third specific example of the carbon porous body is a nitrogen-containing carbon gel. “Nitrogen-containing carbon gel” refers to a carbon gel in which at least the vicinity of the surface is composed of nitrogen-containing carbon. The “surface” includes not only the outer surface but also the inner surface of the pore. The carbon gel whose surface is made of nitrogen-containing carbon has the advantage that the amount of enzyme supported, stability and activity are improved.

The atomic ratio (N / C ratio) between N atoms and C atoms in the nitrogen-containing carbon gel is preferably 0.01 or more. When the N / C ratio is less than 0.01, N atoms are reduced and the number of adsorption sites capable of interacting with the enzyme is reduced. The N / C ratio is more preferably 0.05 or more.
On the other hand, the N / C ratio is preferably 0.4 or less. When the N / C ratio exceeds 0.4, the strength of the carbon skeleton decreases, and it becomes difficult to maintain the pore structure. The N / C ratio is more preferably 0.3 or less.
The N / C ratio in such nitrogen-containing carbon can be obtained by CHN elemental analysis or XPS. Moreover, since the other point regarding nitrogen-containing carbon gel is the same as that of carbon gel, description is abbreviate | omitted.

The enzyme that is an electrode catalyst is supported on the surface of the carrier. “The surface of the carrier” refers to the outer surface of the carrier and the inner surface of the pores. It is preferable that at least a part of the enzyme is supported on the inner surface of the pore. The larger the amount of enzyme supported in the pore, the better.
The amount of the enzyme supported on the carrier is not particularly limited as long as the enzyme activity is shown. In general, the higher the amount of enzyme supported, the higher the activity. In order to obtain a practically sufficient activity, the amount of the enzyme supported is preferably 0.01 parts by mass or more with respect to 100 parts by mass of the carbon porous body. On the other hand, when the amount of the enzyme loaded becomes excessive, the effect is saturated and there is no actual benefit. Therefore, the amount of the enzyme supported is preferably 80 parts by mass or less with respect to 100 parts by mass of the carbon porous body.

The enzyme electrode according to the present invention may be composed only of the carrier and the enzyme described above, or may further carry a mediator that promotes the transfer of electrons between the carbon porous body and the enzyme. When a mediator is further supported on the surface of the carrier, more efficient electron conduction is expected.
Specific examples of mediators include metal cyanides such as ABTS and WCN, and Os complexes. These mediators are preferably immobilized on the electrode surface, and for this purpose, it is desirable to bind to a polymer such as poly-1-vinylimidazole and insolubilize it. Furthermore, the mediator may be directly immobilized on the carbon support. These mediators may be used alone or in combination of two or more.

  When the carrier is in the form of a sheet, the enzyme electrode according to the present invention can be used in a state where an enzyme (and a mediator if necessary) is carried on the surface of the electron conductive carrier. On the other hand, when the carrier is in the form of powder, the carrier carrying the enzyme can be further used by fixing it on the surface of a suitable metal electrode (for example, Pt electrode).

Next, the manufacturing method of the carbon porous body used for a support | carrier is demonstrated. Among the carbon porous bodies, mesoporous carbon, carbon gel, and nitrogen-containing carbon gel can be produced by the following method.
[1. Method for producing mesoporous carbon]
The method for producing mesoporous carbon particles is not particularly limited, and can be produced, for example, by the following method. That is, porous molecules (templates) such as silica and titania with regularly arranged mesopores are adsorbed and impregnated with organic molecules such as sucrose and furfuryl alcohol, and then carbonized under an inert atmosphere. Thereafter, when the template is dissolved and removed with hydrofluoric acid, NaOH / EtOH or the like, mesoporous carbon particles are obtained. As a template, for example, silica mesoporous material MCM-48 can be used.

[2. Method for producing carbon gel]
The manufacturing method of carbon gel is not specifically limited, For example, it can manufacture by the following methods.
That is, first, an organic gel is synthesized according to the method described in the literature (RWPekala, CTAlviso, FMKong, and SSHulsey, J. Non-cryst. Solids, vol. 145, p. 90 (1992)). That is, a phenol resin such as resorcinol and an aldehyde such as formaldehyde are reacted in the presence of an alkali catalyst or an acid catalyst, and aged to obtain an organic gel composed of the phenol resin. Next, after drying the obtained organic gel, the carbon gel can be obtained by baking and carbonizing in an inert atmosphere.

[3. Method for producing nitrogen-containing carbon gel]
The method for producing the nitrogen-containing carbon gel is not particularly limited, and for example, it can be produced by the following method.
(A) A method of introducing N atoms into carbon gel using nitric oxide.
The introduction of N atoms into the carbon gel can be performed, for example, according to the method described in the literature (P.Chambrion et al., Energy & Fuels vol.11, p.681-685 (1997)). is there. That is, a carbon gel is placed in a quartz reaction tube and heated to about 950 ° C. under a helium stream. Thereafter, NO diluted with helium (concentration of about 1000 ppm) is introduced into the reaction tube and reacted at a reaction temperature of about 600 to 900 ° C. The reaction time required for this reaction is not particularly limited, but if the reaction time is extended, the amount of nitrogen incorporated into the carbon skeleton increases.

(B) A method of depositing nitrogen-containing carbon on the surface of the carbon gel by a thermal CVD method.
In this method, a nitrogen-containing organic compound is introduced into the pores of the carbon gel, and the nitrogen-containing carbon is deposited on the surface of the carbon gel by thermally decomposing the nitrogen-containing organic compound. That is, first, a carbon gel is placed in a reaction tube and heated to a predetermined temperature while introducing an inert gas such as nitrogen or argon into the reaction tube. Next, a nitrogen-containing organic compound in a gaseous state is introduced into the reaction tube while maintaining the heated state, thereby performing a CVD reaction for a predetermined time while introducing the nitrogen-containing organic compound into the pores of the carbon gel. As a result, nitrogen-containing carbon having a skeleton formed of C atoms and N atoms can be deposited in the pores of the carbon gel. The precipitation step by the thermal CVD method is usually performed in an inert atmosphere such as nitrogen or argon because C combustion occurs when the reaction atmosphere is an oxidizing atmosphere.

The nitrogen-containing organic compound used here is not particularly limited as long as it is an organic compound containing an N atom, and examples thereof include nitrogen-containing heterocyclic compounds, amines, imines, and nitriles. Examples of the nitrogen-containing heterocyclic compound include a nitrogen-containing heterocyclic monocyclic compound and a nitrogen-containing condensed heterocyclic compound.
Nitrogen-containing monocyclic compounds include pyrrole and its derivatives as 5-membered ring compounds, diazoles and derivatives thereof such as pyrazole and imidazole, and pyridine and its derivatives as 6-membered ring compounds, pyridazine, pyrimidine and pyrazine, etc. Examples thereof include diazines and derivatives thereof, triazines, and triazine derivatives such as melamine and cyanuric acid.
Examples of the nitrogen-containing condensed heterocyclic compound include quinoline, phenanthroline, and purine.

Examples of amines include primary to tertiary amines, diamines, triamines, polyamines, and amino compounds.
Examples of primary to tertiary amines include aliphatic amines such as methylamine, ethylamine, dimethylamine and trimethylamine, aromatic amines such as aniline, and derivatives thereof.
Examples of amino compounds include amino alcohols such as ethanolamine.
Examples of imines include pyrrolidine and ethylimine.
Examples of nitriles include aliphatic nitriles such as acetonitrile and aromatic nitriles such as benzonitrile.
Other nitrogen-containing organic compounds include polyamides such as nylon, amino sugars such as galactosamine, nitrogen-containing polymer compounds of polyacrylonitrile sugar, amino acids and polyimides.

In the precipitation step by the thermal CVD method, when the nitrogen-containing organic compound is in a liquid state at room temperature, the nitrogen-containing organic compound can be introduced into the reaction tube as a gas state by vapor evaporation using a bubbler, a mass flow pump or the like. . At this time, it is preferable to introduce a nitrogen-containing organic compound in a gaseous state using nitrogen, argon or the like as a carrier gas. Furthermore, it is preferable to prevent backflow by installing a bubbler containing liquid paraffin or the like on the reaction tube outlet side so that the gas once circulated in the reaction tube does not flow back from the reaction tube outlet side.
When the nitrogen-containing organic compound is in a solid state at normal temperature, a heating evaporator (sublimation) can be installed on the reaction tube inlet side, and the nitrogen-containing organic compound can be introduced into the reaction tube as a gas state by heating. Moreover, the temperature of the evaporator at this time needs to be adjusted to a temperature at which the nitrogen-containing organic compound is not thermally decomposed.

When the nitrogen-containing organic compound has polymerizability, it can be polymerized in advance in the pores of the carbon gel and then thermally decomposed in an inert atmosphere in a reaction tube.
Further, when the nitrogen-containing organic compound is not vaporized by heating, the nitrogen-containing organic compound is previously introduced into the pores of the carbon gel by a solution adsorption method, an evaporation to dryness method, etc. By carrying out thermal decomposition below, nitrogen-containing carbon can be deposited in the pores of the carbon gel.

  The reaction temperature in the precipitation step by the thermal CVD method is not particularly limited as long as the nitrogen-containing organic compound is thermally decomposed and carbonized, but is preferably 300 to 1000 ° C. The reaction temperature is more preferably 500 to 700 ° C. When the reaction temperature is less than 300 ° C., thermal decomposition of the nitrogen-containing organic compound is difficult to occur, so that the deposition rate of nitrogen-containing carbon is slowed, and the reaction time and energy consumption tend to increase. On the other hand, when the reaction temperature exceeds 1000 ° C., it is difficult for carbon to remain in the carbon skeleton, so the N / C ratio decreases.

In the precipitation step, the amount of nitrogen-containing carbon deposited in the pores of the carbon gel is not particularly limited, but when the specific surface area per 1 g of the carbon gel is Ym ² , it is (0.0001 × Y) g or more. Preferably there is. When the amount of nitrogen-containing carbon deposited is less than (0.0001 × Y) g, the amount of deposited is small, and thus there is a possibility that the adsorptive improvement by N atoms cannot be obtained.

  The amount of precipitation correlates with the CVD reaction time, and the amount of precipitation can be controlled by adjusting the CVD reaction time. Furthermore, the amount of precipitation varies depending on the CVD reaction temperature, the type of carbon gel, the type of nitrogen-containing organic compound, the flow rate when introducing the nitrogen-containing organic compound, etc., but the CVD reaction time is appropriately adjusted in each case. By doing so, the amount of precipitation can be adjusted.

Next, the method for producing the enzyme electrode according to the present invention will be described.
First, an enzyme is supported on the surface of the carbon porous body. The enzyme loading method is not particularly limited, and various methods such as a sublimation method and an impregnation method can be used, but an impregnation method is preferable.

Enzyme loading by the impregnation method is performed as follows. That is, first, the enzyme is dissolved in water or a buffer so as to have a concentration at which precipitation does not occur (preferably, 0.1 to 1000 mg / ml). Then, the powder carrier is suspended at a temperature (preferably 0 to 50 ° C.) at which the solution does not freeze and the enzyme is not denatured, or the sheet carrier is immersed, and the enzyme and carrier Contact. The contact time is preferably at least 5 minutes or more, more preferably 30 minutes or more. As a result, the enzyme is immobilized on the surface or pores of the carrier.
The concentration at which the carrier is suspended in the solution is not particularly limited, but is preferably about 0.1 to 1000 mg / ml. Moreover, you may have the process of isolate | separating and taking out a support | carrier from a solution by performing centrifugation etc. after the carrying | support process. Alternatively, the liquid component may be removed by drying or the like.

  As a method for supporting the mediator on the carrier, a method similar to the method for supporting the enzyme on the carrier can be used. Further, the enzyme and the electron transfer medium may be simultaneously supported on the carrier.

Next, the operation of the electrode catalyst and the enzyme electrode according to the present invention will be described.
FIG. 1 shows the molecular structure of CueO derived from E. coli, which is a kind of multi-copper oxidase (Sue A. Roberts et al., PNAS, vol. 99 (2002), 2766-2771). As shown in FIG. 1, CueO has mononuclear blue copper (type ICu) functioning as an electron transfer gate and trinuclear copper clusters (type IICu, type IIICu) serving as active centers in the three-dimensional structure of the protein. Yes, mononuclear blue copper and trinuclear copper clusters are in different openings, respectively (Kunishige Kataoka, Biochemistry, Vol. 77 (2005), pp. 148-153). In addition, the two openings are respectively on the front and back surfaces of the three-dimensional structure formed by the protein. Furthermore, natural type CueO has mononuclear blue copper covered with helical molecules called helix 5 at the top.

When such CueO is used as an electrode catalyst for an enzyme electrode, a higher current density can be obtained compared to a conventional oxidoreductase.
Although the details of the reason are unknown, it is thought to be due to the following reasons.
(1) Since CueO separates the active center that catalyzes the oxidation reaction and the electron transfer gate in the enzyme molecule, even if the enzyme is immobilized on the electrode solid surface, the catalytic reaction and the electron transfer can be achieved simultaneously.
(2) Since CueO has more electron transfer gates on the surface of the molecule, the distance from the electrode is reduced in the state of being fixed on the solid surface, and the efficiency of electron transfer is improved.

Moreover, when fixing CueO to a support | carrier, when a carbon porous body which has a predetermined pore diameter, pore distribution, and specific surface area is used, a high current density is obtained.
this is,
(1) Since most of the enzyme can be fixed in the pores of the carbon porous body, the electron transfer between the carrier / enzyme is facilitated, and
(2) Since the change in the three-dimensional structure of the protein is suppressed by the pore wall, the deactivation due to heat is reduced and the thermal stability is improved.
it is conceivable that.
Further, when carbon gel is used as the carbon porous body, high electron transfer efficiency can be obtained. This is considered to be due to having a large amount of pores suitable for enzyme loading, and forming a three-dimensional network in which the pores are interconnected.
Furthermore, when nitrogen-containing carbon gel is used as the carbon porous body, high electron transfer efficiency can be maintained over a long period of time. This is presumably because the introduction of nitrogen into the carbon gel introduces polar groups into the pore inner walls, and new bonds such as hydrogen bonds occur with the hydrophilic groups on the protein surface.

(Example 1, Comparative Examples 1 and 2)
[1. Test method]
E. coli-derived CueO (Example 1), Urushi-derived laccase (Comparative Example 1), or Myrotheciumu sp.-derived bilirubin oxidase (BOD) (Comparative Example 2) is dissolved in phosphate buffer (0.05 M, pH 7.0). 8 μM enzyme solution was prepared.
Using the above enzyme solution saturated with oxygen in advance, a cyclic voltamgram method (potential: −50 to 650 mV, sweep speed: 20 mV / sec) using a HOPG (highly oriented pyrolytic graphite) electrode as a working electrode, The direct electron transfer characteristics between enzyme and electrode were evaluated. A platinum electrode was used as the counter electrode, and a silver / silver chloride electrode was used as the reference electrode.

[2. result]
FIG. 2 shows a cyclic voltamgram. From FIG. 2, the oxidation current density of 0 V in the quiescent state of laccase (Comparative Example 1) and BOD (Comparative Example 2) is −500 μA / cm ² or less, whereas the oxidation of 0 V in the quiescent state of CueO from Escherichia coli. It can be seen that the current density is about −2000 μA / cm ² .

(Examples 2 and 3, Comparative Examples 3 and 4)
[1. Preparation of carbon gel]
Resorcinol 5.5 g (Wako Pure Chemical Industries) and sodium carbonate 26.5 mg (Wako Pure Chemical Industries, Ltd.) were dissolved in 16.9 g of distilled water, and then a 37% formaldehyde solution 8.1 g (Wako Pure Chemical Industries) was added and mixed with stirring. The mixed solution became light yellow and transparent. In addition, the molar ratio of each component is resorcinol: sodium carbonate: formaldehyde = 200: 1: 400.
Next, water was added to the obtained undiluted | stock solution, and it diluted by 2 times by volume ratio. The diluted solution was put in a vial and sealed, and allowed to stand at room temperature for 24 hours, at 50 ° C. for 24 hours, and further at 90 ° C. for 72 hours to obtain a hydrated organic gel.

Next, in order to remove the moisture in the organic gel, the organic gel was immersed in acetone (Wako Pure Chemical) as an exchange solvent. When the organic gel is immersed in acetone, the moisture in the organic gel diffuses into the acetone, and the moisture in the gel can be replaced with acetone. When the diffusion of moisture was saturated, the operation of replacing the acetone with a new one was repeated several times to completely replace the moisture in the gel with acetone. Next, the immersion solvent was changed to n-pentane (Wako Pure Chemical Industries), and the solvent exchange-immersion was repeated until acetone in the organic gel was completely replaced with n-pentane. Furthermore, the organic gel solvent-substituted with n-pentane was air-dried to obtain a dried organic gel.
The obtained dried organic gel was heated at 1000 ° C. under a nitrogen stream (flow rate: 300 ml / min) to carbonize the organic gel. The heating time was 6 hours.

[2. Preparation of enzyme electrode]
A slurry in which 6.7% carbon gel (Example 2) or Ketjen Black (Example 3) is suspended in an NMP (N-Methyl-pyrrolidone) solution containing 5% PDVF (Polyviniylidine Difluoride) is prepared. The slurry was added to the surface of a glassy carbon electrode having a diameter of 6 mm (manufactured by BAS, model number: 002012) and spin-coated (3000 rpm) to coat the surface of the electrode with a carbon support. The obtained carbon-modified electrode was immersed in an E. coli-derived CueO aqueous solution (12.5 mg / ml) at 4 ° C. overnight to immobilize the enzyme on the carbon-modified electrode.
Further, according to the same procedure, urushi-derived laccase was immobilized on a carbon gel (Comparative Example 3) or ketjen black (Comparative Example 4) modified electrode.

[3. Test method]
[2. The electrical characteristics of the electrodes were evaluated by the cyclic voltamgram method (electric potential: current sweep between 200 to 500 mV, sweep speed 20 mV / sec) using the various enzyme electrodes obtained in the above. Platinum was used for the counter electrode, and a silver / silver chloride electrode was used for the reference electrode. As the electrolyte, a 50 mM potassium phosphate buffer solution (pH 7.0) saturated with oxygen was used. Furthermore, the electrical characteristics were evaluated in a state where both the electrolytic solution and the enzyme electrode were stationary.

[4. result]
FIG. 3 shows a cyclic voltamgram. From FIG. 3, it can be seen that the amount of current increases for both laccase and CueO in the carbon gel as compared with ketjen black. In particular, in the CueO / CG combination, the current density at 200 mV increased about 7 times compared to laccase / KB.

  Although the embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments, and various modifications can be made without departing from the scope of the present invention.

The electrode catalyst according to the present invention can be used as an electrode catalyst for various electrochemical devices such as biosensors and fuel cells, and solar cells.
The enzyme electrode according to the present invention can be used as an electrode for various electrochemical devices such as biosensors and fuel cells, and solar cells.

It is the molecular structure of Escherichia coli-derived CueO, which is a kind of multi-copper oxidase. It is a cyclic voltamgram of the enzyme solution containing various multi copper oxidase. It is a cyclic voltagram of an enzyme electrode containing various multi-copper oxidases supported on various carbon carriers.

Claims (4)

An electrode catalyst comprising CueO derived from E. coli . Carbon porous body,
An enzyme electrode comprising: an electrocatalyst made of Escherichia coli-derived CueO supported on the surface of the carbon porous body. The carbon porous body, the following (i) and (ii) satisfying carbon gel der Ru enzyme electrode according to claim 2.
(A) No X-ray diffraction peak is observed in the scan region 2θ = 0.5 to 10 ° (CuKα ray).
(B) In the pore size distribution calculated from the adsorption / desorption isotherm,
When the pore diameter value (d) at the distribution peak top is in the range of 2 nm or more and less than 10 nm, 60% or more of the total pore volume in the pore diameter region of d ± 2 nm with respect to the pore diameter value (d). Is included,
When the pore diameter value (D) at the distribution peak top is in the range of 10 nm to 50 nm, (0.75 × D) to (1.25 × D) nm with respect to the pore diameter value (D). 60% or more of the total pore volume is included in the pore diameter region. The enzyme electrode according to claim 2 or 3, further comprising a mediator that promotes transfer of electrons between the carbon porous body and the CueO.

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